Brain Tumor Biology and Etiology
Brain tumors are considered to have one of the least favorable prognoses for long term survival: the average life expectancy of an individual diagnosed with a central nervous system (CNS) tumor is just eight to twelve months. Several unique characteristics of both the brain and its particular types of neoplastic cells create daunting challenges for the complete treatment and management of brain tumors. Among these are 1) the physical characteristics of the intracranial space, 2) the relative biological isolation of the brain from the rest of the body, 3) the relatively essential and irreplaceable nature of the organ mass, and 4) the unique nature of brain tumor cells.
First and foremost, the intracranial space and physical layout of the brain create significant obstacles to treatment and recovery. The brain is made of, primarily, astrocytes (which make up the majority of the brain mass, and serve as a scaffold and support for the neurons), neurons (which carry the actual electrical impulses of the nervous system), and a minor contingent of other cells such as insulating oligodendrocytes (which produce myelin). These cell types give rise to primary brain tumors (e.g., astrocytomas, neuroblastomas, glioblastomas, oligodendrogliomas, etc.) Although the World Health Organization has recently established standard guidelines, the nomenclature for brain tumors is somewhat imprecise, and the terms astrocytoma and glioblastoma are often used broadly. The brain is encased in the relatively rigid shell of the skull, and is cushioned by the cerebrospinal fluid, much like a fetus in the womb. Because of the relatively small volume of the skull cavity, minor changes in the volume of tissue in the brain can dramatically increase intracranial pressure, causing damage to the entire organ (i.e., “water on the brain”). Thus, even small tumors can have a profound and adverse affect on the brain's function. In contrast, tumors in the relatively distensible abdomen may reach several pounds in size before the patient experiences adverse symptoms. The cramped physical location of the cranium also makes surgery and treatment of the brain a difficult and delicate procedure. However, because of the dangers of increased intracranial pressure from the tumor, surgery is often the first strategy of attack in treating brain tumors.
In addition to its physical isolation, the brain is chemically and biologically isolated from the rest of the body by the so-called “Blood-Brain-Barrier” (or BBB). This physiological phenomenon arises because of the “tightness” of the epithelial cell junctions in the lining of the blood vessels in the brain. Although nutrients, which are actively transported across the cell lining, may reach the brain, other molecules from the bloodstream are excluded. This prevents toxins, viruses, and other potentially dangerous molecules from entering the brain cavity. However, it also prevents therapeutic molecules, including many chemotherapeutic agents that are useful in other types of tumors, from crossing into the brain. Thus, many therapies directed at the brain must be delivered directly into the brain cavity (e.g., by an Ommaya reservoir), or administered in elevated dosages to ensure the diffusion of an effective amount across the BBB.
With the difficulties of administering chemotherapies to the brain, radiotherapy approaches have also been attempted. However, the amount of radiation necessary to completely destroy potential tumor-producing cells also produce unacceptable losses of healthy brain tissue. The retention of patient cognitive function while eliminating the tumor mass is another challenge to brain tumor treatment. Neoplastic brain cells are often pervasive, and travel throughout the entire brain mass. Thus, it is impossible to define a true “tumor margin,” unlike, for example, in lung or bladder cancers. Unlike reproductive (ovarian, uterine, testicular, prostate, etc.), breast, kidney, or lung cancers, the entire organ, or even significant portions, cannot be removed to prevent the growth of new tumors. In addition, brain tumors are very heterogeneous, with different cell doubling times, treatment resistances, and other biochemical idiosyncrasies between the various cell populations that make up the tumor. This pervasive and variable nature greatly adds to the difficulty of treating brain tumors while preserving the health and function of normal brain tissue.
Although current surgical methods offer considerably better post-operative life for patients, the current combination therapy methods (surgery, low-dosage radiation, and chemotherapy) have only improved the life expectancy of patients by one month, as compared to the methods of 30 years ago. Without effective agents to prevent the growth of brain tumor cells that are present outside the main tumor mass, the prognosis for these patients cannot be significantly improved. Although some immuno-affinity agents have been proposed and tested for the treatment of brain tumors, see, e.g., the tenascin-targeting agents described in U.S. Pat. No. 5,624,659, these agents have not proven sufficient for the treatment of brain tumors. Thus, therapeutic agents which are directed towards new molecular targets, and are capable of specifically targeting and killing brain tumor cells, are urgently needed for the treatment of brain tumors.
ARP-2 (Angiopoeitin Related Protein-2, Angiopoeitin Like-2 [ANGPTL-2])
Angiopoeitin related protein-2 (ARP-2), is related to the angiopoeitin family of proteins, that includes Ang-1 and Ang-2. Like members of the angiopoeitin family, ARP-2 contains a coiled-coil domain in the amino terminal portion and a fibrinogen-like domain in the carboxyl terminal portion. However, ARP-2 has a low homology with Ang-1 and Ang-2 and unlike Ang-1 and Ang-2, ARP-2 does not bind to the Tie-2 receptor, nor does ARP-2 bind to the closely related Tie-1 receptor. Hence, ARP-2 is believed to be part of a newly identified family of proteins termed angiopoeitin related proteins. Like the angiopoeitins, ARP-2 is a member of the fibrinogen superfamily, which also includes the fibrinogens and lectins.
ARP-2 is a glycosylated, secretory protein that induces sprouting in endothelial cells, most likely through autocrine or paracrine signaling, and it is preferentially expressed in the blood vessels and muscle cells. Hence, ARP-2 mediates the differentiated state of endothelial cells or for vascular remodeling and development. ARP-2 has not heretofore been associated with brain tumors.
SPARC (Secreted Protein, Acidic, Cysteine-rich; Osteonectin, Basement Membrane Protein (bm) 40)
Secreted protein acidic and rich in cysteine, SPARC or BM-40, is a member of the counter-adhesive family of proteins. It is a developmentally regulated, secreted glycoprotein expressed in fetal astrocytes, particularity during tissue remodeling, vessel morphogenesis, and in response to stress. It has been hypothesized that SPARC may affect cell migration and vascular morphogenesis either by directly interacting with extracellular matrix (ECM) proteins (such as collagens I, III, IV and V) or by initiating a receptor mediated signaling event that induces changes in cytoplasmic components associated with focal adhesions. SPARC has been found to bind directly to vitronectin, a multifunctional adhesive protein that is a component of the brain vascular basement membranes.
SPARC may indirectly affect cell migration and motility by regulating the expression of matrix metallo-proteases and by modulating the expression of other proteolytic enzymes (such as collagenase) that degrade the ECM. Increased SPARC expression has also been observed in two forms of low-grade malignant gliomas, in all grades of human astrocytic tumors, and in tumor cells invading adjacent brain at the tumor/brain interface. Hence, SPARC may be an astrocytoma invasion related gene that functions in connection with vitronectin to balance the modulation of cellular adhesion to the ECM and it may promote diffuse tumor cell infiltration into adjacent brain by affecting both tumor and endothelial cell-ECM interactions.
Because SPARC is also found in bone, dentine, and many normal and neoplastic human soft tissues it may also play a regulatory function in the control of such diverse processes as bone mineralization, cell shape, tissue remodeling or repair, cell migration, proliferation, and differentiation. SPARC is also synthesized, stored, and secreted by human blood platelets, binds to plasminogen, and enhances tissue plasminogen activator conversion of plasminogen to plasmin.
c-MET (Met Proto-oncogene Tyrosine Kinase, Hepatocyte Growth Factor Receptor [HGFR])
c-MET is a member of the Hepatocyte Growth Factor Receptor (HGFR) family and a heterodimeric cellular receptor for Hepatocyte Growth Factor (HGF). c-MET contains a disulfide-linked α-chain of 50-kDa (which is located in the extracellular domain,) a 145-kDa β-chain (which includes an extracellular region,) a transmembrane spanning domain, and an intracellular tyrosine kinase domain that can be activated by autophosphorylation. Hence, HGFR is a subset of the protein tyrosine-kinase family of membrane-spanning, cell surface receptors.
The receptor-ligand pair, c-MET and HGF, function as a growth factor, regulating cell growth, migration, and morphogenesis, and hence, may play a role in neoplastic formation and metastasis. Upon HGF or macrophage stimulating protein (MSP) binding, the c-MET protein receptor goes through a conformational change wherein the intracellular tyrosine residues of the β subunit become phosphorylated at residue 1235, and a second messenger signal cascade is induced. This change activates c-MET's intracellular receptor kinase activity, which is important to the growth and differentiation of epithelial cells in normal and malignant tissues. c-MET has been identified in both normal brain and on glial tumors, and is thought to be determinant in the pathological processes of various malignancies. For instance, detailed studies have shown that glioblastoma multiforme (GBM), a highly malignant brain tumor of astrocytic origin, expresses c-MET, and this research suggests a role in tumor progression.
BEHAB (Brain-enriched Hyaluronan Binding Protein, Brevican)
BEHAB is a brain-specific, extracellular matrix protein, that is a member of the chondroitin sulfate proteoglycan (CSPG) family. BEHAB is expressed only in the CNS. Although its function is unclear, BEHAB is reported to bind to HA at the N-terminus, lectins at the C-terminus, and may mediate binding of other ECM components like tenascin. This suggests that BEHAB may play a role in cell-cell and cell-matrix interactions thereby maintaining the extracellular environment of the brain. It has been reported that the highest levels of expression of BEHAB is during brain development and at times and places where glial cells are highly motile, as in cases of brain injury or trauma. BEHAB expression is also unregulated in primary gliomas of the central nervous system, but not in tumors of non-glial origin. In surgical samples of human gliomas (including astrocytoma, oligodendroglioma, and glioblastoma tumors), BEHAB expression is consistently and dramatically increased over the level of expression in the normal brain. Hence, BEHAB expression correlates with an invasive phenotype that promotes gliogenesis by contributing to cell movement through the ECM.
CD-44 Antigen
CD-44 is a single-path, type I transmembrane protein with extracellular domains that are flexibly linked to the transmembrane segment. CD-44 is a member of the cartilage link protein family and belongs to the hyaloadherin or link protein superfamily (LPSF). As other members of the LPS family, CD-44 can be extensively glycosylated and is typically decorated with glycosaminoglycans (e.g., chondroitin, heparin, and keratin sulfate). The genomic structure of CD-44 consists of 21 exons, at least 11 of which can be variably spliced (v1–v10), that are located in the membrane-proximal extracellular region. Alternative splicing of these exons give rise to a variety of CD-44 isoforms (at least 30 different isoforms have been characterized to date) that are widely distributed and expressed in a cell-specific manner. Among the most frequently occurring isoforms are CD-44H, expressed on hematopoietic cells, and CD-44E, expressed in epithelial cells. CD-44(H) has also been found to be expressed in lymphocytes, macrophages, erythrocytes, fibroblasts, epithelial and endothelial cells, and neurons. It is the predominant isoform in normal brain and neuroectoderm-derived tumors and is expressed on both normal astrocytes and oligodendrocytes as well on neoplastic astrocytes and glioblastomas.
The family of CD-44 proteins has been implicated in lymphocyte activation and homing, endothelial migration, and tumor cell metastasis. CD-44 is believed to be the major receptor for Hyaluronic acid (HA). CD-44/HA interactions underlie a wide spectrum of functions in embryonic morphogenesis and organogenesis, hematopoeisis, lymphocyte homing. CD-44 also mediates the attachment of glioma cells to chondroitin sulfate, types I and IV collagen, fibronectin laminin, vitronectin and Martrigel. This suggest that CD-44 may play a role in cell-cell and cell-matrix interactions, affecting the extracellular environment of the brain. Because HA is a major component of the brain ECM, and CD-44 is one of the principal cellular receptors of HA, CD-44 expression coincides with brain tumor growth and invasiveness.
PTN (Pleiotrophin, Heparin Binding Growth Factor 8, Neurite Growth-promoting Factor 1)
Pleiotrophin or PTN, is a platelet-derived, growth factor inducible, member of the pleiotrophin family of proteins that includes midkine and retinoic acid-induced heparin-binding protein. It is a developmentally regulated, secreted cytokine that stimulates mitogenesis, angiogenesis, and neurite and glial process outgrowth guidance activities. During development PTN is expressed in the brain, intestine, muscle, skin, heart, lung and kidney. In the adult, PTN is found primarily in the brain in association with axonal tracts during active mitogenesis and may therefore play an important role in the development and maintenance of the nervous system. It has been found to bind heparin, heparin sulfate proteoglycans, the extracellular matrix, and is also a natural ligand for receptor protein tyrosine phosphatase (RPTP), signaling through ligand dependant receptor inactivation of RPTP. Receptor mediated endocytosis occurs following PTN binding and may be disrupted by heparin.
PTN has also been found to have oncogenic properties, inducing malignant transformation and tumor growth and progression. It has been described as a proto-oncogene that is expressed in many human tumors and cell lines derived from human tumors. PTN is a mitogen for fibroblasts, epithelial and endothelial cells, stimulates plasminogen-activator production, can induce tube formation, and therefore can serve as a tumor angiogenesis factor.
OPN (Osteopontin, Secreted Phosphoprotein 1, Bone Sialoprotein-1)
Osteopontin or OPN, is a member of the osteopontin family. It is a glycosylated sialoprotein that is heavily phosphorylated and expressed in a variety of cells including bone, kidney, placenta, nerve cells and macrophages, as well as T lymphocytes, epidermal and bone cells. OPN is a part of the mineralized bone matrix and may play a role in bone resorption, by facilitating the attachment of osteoclasts to the bone surface, and may be functionally important as an adhesive and chemotactic molecule for vascular cells. OPN is a secreted protein that binds tightly to hydroxyapatite, and hence, is important to cell matrix interactions. It has been observed to interact with the CD-44 homing receptor to physiologically induce macrophage chemotaxis, which may be a mechanism utilized by metastatic brain tumors in the process of dissemination.
OPN has been observed in the microvasculature of glioblastomas associated with VEGF expression and OPN mRNA has been found to be overexpressed in high grade and metastatic brain tumors. Hence, OPN expression correlates with the malignancy grade of gliomas.
VIPR-2 (Vasoactive Intestinal Peptide Receptor-2)
Vasoactive intestinal polypeptide receptor II (VIPR-2), VPAC-2, is a member of the G-protein receptor family, which includes such members as the calcitonin, parathyroid hormone, secretin, glucagon and VIP-1 receptors. VIPR-2 is a seven-transmembrane spanning G protein-coupled receptor that responds to VIP by stimulating cAMP production. VIPR-2 is found in the brain as well as peripheral tissues such as the pancreas, skeletal muscle, heart, lung, kidneys, stomach, adipocytes and the liver, and in various cells of the immune system. In the brain, VIPR-2 functions as a neuroendocrine hormone and neurotransmitter receptor, and is found in the thalamus, hippocampus, suprachiasmatic nucleus and hypothalamus.
VIPR-2 is encoded by a nucleotide sequence of approximately 2.8 kb, which codes for a 438 amino acid sequence of approximately 48–64 kDa. The receptor-ligand pair, VIPR-2 and VIP, have various functions dependent upon the tissue where in they are located. VIP is a late-developing, 28 amino acid peptide that, along with its receptor, is widely distributed throughout the peripheral body, and plays a role in cardiovascular, reproductive, pulmonary, immune and gastrointestinal systems, to effect vasodilatation, bronchodilation, immunosuppression, hormonal secretion, and increased gastric motility. However, the cerebral cortex has one of the highest reported concentrations of VIP, localized to intrinsic neurons throughout all neocortical regions. In the brain, VIP and its receptor, have behavioral, electrophysiological, secretory, metabolic, vascular, and mitogenic effects. For instance, the receptor-ligand pair play a role in cortical differentiation, the relaying of sensory information to the cortex, and the regulation of morphogenic events by the release of diffusible signals from glial cells. VIPR-2 and VIP also play a role in the growth and differentiation of neuroblastomas.
TSPAN3 (Tetraspanin 3, Tetraspanin TM-4A)
The Tetraspanin superfamily, is a family of approximately 20 integral membrane proteins that are broadly expressed in most human tissues including neural and bone marrow derived tissues. The family shares a common motif that includes four putative transmembrane domains (TM1–4), a small extracellular domain (EC1) of 20–27 amino acids, and a larger extracellular domain (EC2) between TMS3 and TMS4 of 70–130 amino acids. Two conserved features of tetraspanins are critical to their structure and function. First, charged residues are present in or near the TM domains, second, a cluster of cysteine residues is in the putative EC2 domain. Most of the tetraspanins are modified by N-glycosylation.
Many Tetraspanin proteins affect the regulation of cellular proliferation, motility, differentiation, development. In some cells, Tetraspanins may act as adapters in ultimeric complexes that link plasma membrane proteins, like integrins, into signaling complexes with other signaling molecules (e.g., phosphatidylinositol 4-kinase) at the plasma membrane and play a role in integrin-mediated cell migration, metastasis and tumor cell invasion. A number of tetraspanins have also been discovered as tumor-associated proteins, including C-029, PETA-3/SFA-1, and SAS, which is amplified in a subset of sarcomas. Of the various TM4SF proteins, CD9, CD63, CD81, CD82, and CD151 are the most widely distributed. CD9 is expressed on 90% of non-T cell acute lymphoblastic leukemia cells and on 50% of chronic lymphocytic and acute myeloblastic leukemias. CD63 is also expressed in early stage melanomas.
Protein Tyrosine Phosphatase Receptor Zeta (PTPζ)
Vital cellular functions, such as cell proliferation and signal transduction, are regulated in part by the balance between the activities of protein kinases and protein phosphatases. These protein-modifying enzymes add or remove a phosphate group from serine, threonine, or tyrosine residues in specific proteins. Some tyrosine kinases (PTK's) and phosphatases (PTPase's) have been theorized to have a role in some types of oncogenesis, which is thought to result from an imbalance in their activities. There are two classes of PTPase molecules: low molecular weight proteins with a single conserved phosphatase domain such as T-cell protein-tyrosine phosphatase (PTPT; MIM 176887), and high molecular weight receptor-linked PTPases with two tandemly repeated and conserved phosphatase domains separated by 56 to 57 amino acids. Examples of this latter group of receptor proteins include: leukocyte-common antigen (PTPRC; MIM 151460) and leukocyte antigen related tyrosine phosphatase (PTPRF; MIM 179590).
Protein tyrosine phosphatase zeta (PTPζ) [also known as PTPRZ, HPTP-ZETA, HPTPZ, RPTP-BETA(β), or RPTPB] was isolated as a cDNA sequence by two groups in the early nineties. The complete cDNA sequence of the protein is provided in SEQ ID NO. 1, and the complete deduced amino acid sequence is provided in SEQ ID NO. 2. Splicing variants and features are indicated in the sequences. Levy et al. (“The cloning of a receptor-type protein tyrosine phosphatase expressed in the central nervous system” J. Biol. Chem. 268: 10573–10581, (1993)) isolated cDNA clones from a human infant brain step mRNA expression library, and deduced the complete amino acid sequence of a large receptor-type protein tyrosine phosphatase containing 2,307 amino acids.
Levy found that the protein, which they designated PTP-β (PTPζ), is a transmembrane protein with 2 cytoplasmic PTPase domains and a 1,616-amino acid extracellular domain. As in PTP-γ (MIM 176886), the 266 N-terminal residues of the extracellular domain are have a high degree of similarity to carbonic anhydrases (see MIM 114880). The human gene encoding PTPζ has been mapped to chromosome 7q31.3–q32 by chromosomal in situ hybridization (Ariyama et al., “Assignment of the human protein tyrosine phosphatase, receptor-type, zeta (PTPRZ) gene to chromosome band 7q31.3” Cytogenet. Cell Genet. 70:52–54 (1995)). Northern blot analysis has shown that showed that PTP-zeta is expressed only in the human central nervous system. By in situ hybridization, Levy et al. (1993) localized the expression to different regions of the adult human brain, including the Purkinje cell layer of the cerebellum, the dentate gyrus, and the subependymal layer of the anterior horn of the lateral ventricle. Levy stated that this was the first mammalian tyrosine phosphatase whose expression is restricted to the nervous system. In addition, high levels of expression in the murine embryonic brain suggest an important role in CNS development.
Northern analysis has shown three splice variants: the extracellular proteoglycan phosphacan, which contains the full extracellular region of the protein, and the long (α) and short (β) forms of the transmembrane phosphatase. The β form lacks the extracellular 860 aa long insert domain of the protein, therefore it is not glycosylated. PCR studies of the gene in rat genomic DNA indicated that there are no introns at the putative 5′ and 3′ splice sites or in the 2.6 kb segment which is deleted in the short transmembrane protein. The phosphatases and the extracellular proteoglycan have different 3′-untranslated regions. Additional alternative mRNA splicing is likely to result in the deletion of a 7 amino acid insert from the intracellular juxtamembrane region of both long and short phosphatase isoforms. Simultaneous quantitation of the three major isoforms indicated that the mRNA encoding phosphacan had the highest relative abundance in the CNS while that encoding the short phosphatase isoform was most abundant relative to the other PTPζ variants in the PNS.
PTPζ has only been found to be expressed in the nervous system. By in situ hybridization, it has been localized to different regions of the adult brain, including the Purkinje cell layer of the cerebellum, the dentate gyrus, and the subependymal layer of the anterior horn of the lateral ventricle. High levels of PTPζ have been seen in regions of the brain where there is continued neurogenesis and neurite outgrowth, and it seems to play a role in morphogenesis and plasticity of the nervous system. Phosphacan immunoreactivity has been associated with perineuronal nets around parvalbumin-expressing neurons in adult rat cerebral cortex. Neurons as well as astrocytes have been shown to express phosphacan.
The transmembrane forms of PTPζ are expressed on the migrating neurons especially at the lamellipodia along the leading processes. PTPζ is postulated to be involved in the neuronal migration as a neuronal receptor of pleiotrophin distributed along radial glial fibers. PTPζ has been shown to be highly expressed in radial glia and other forms of glial cells that play an important role during development. The anti-PTPζ staining localizes to the radial processes of these cells, which act as guides during neuronal migration and axonal elongation. The pattern of RPTP-zeta expression has also been shown to change with the progression of glial cell differentiation.
The three splicing variants of RPTP-zeta have been shown to have different spatial and temporal patterns of expression in the developing brain. The 9.5-kb and 6.4-kb transcripts, which encode the α and β transmembrane protein tyrosine phosphatases, were predominantly expressed in glial progenitors located in the subventricular zone. The 8.4-kb transcript, which encodes the secreted chondroitin sulfate proteoglycan phosphacan, was expressed at high levels by more mature glia that have migrated out of the subventricular zone. The three transcripts have also been shown to be differentially expressed in glial cell cultures.
In knockout studies, PTPζ-deficient mice were viable, fertile, and showed no gross anatomical alterations in the nervous system or other organs. Therefore, it was deduced that PTPζ is not essential for neurite outgrowth and node formation in mice. The ultrastructure of nerves of the central nervous system in PTPζ-deficient mice suggests a fragility of myelin. However, conduction velocity was not altered. The normal development of neurons and glia in was thought to indicate that PTPζ function is not necessary for these processes in vivo, or that a loss of PTPζ can be compensated for by other protein tyrosine phosphatases expressed in the nervous system.
Following CNS injury, robust induction of phosphatase forms of PTPζ mRNA has been observed in areas of axonal sprouting, and of both phosphatases and phosphacan mRNAs in areas of glial scarring. This is thought to imply that the encoded proteins and the cell adhesion molecules and extracellular matrix proteins to which they bind may contribute to recovery from injury and perhaps also to the regulation of axonal regrowth in the nervous system. Following peripheral nerve crush, all PTPζ mRNAs, including phosphacan and the phosphatase variants with and without the 21 base insert, were observed to be significantly induced in the distal segments of the sciatic nerve with a time course that correlated well with the response of Schwann cells to this injury.
The extracellular domains of PTP, have been shown to be capable of binding to several cell adhesion molecules. Phosphacan, which is the shortest, secreted form of PTPζ, containing the full extracellular region, previously was designated 3F8 and 6B4 chondroitin sulfate proteoglycan or 3H1 keratin sulfate proteoglycan depending on the glycosylation status. It is synthesized mainly by glia and binds to neurons and to the neural cell adhesion molecules Ng-CAM/L1, NCAM, TAG-1/axonin-1, to tenascin-C and R, to amphoterin and pleiotrophin/heparin-binding growth-associated molecule (HB-GAM) (amphoterin and pleiotrophin are heparin-binding proteins that are developmentally regulated in brain and functionally involved in neurite outgrowth). Binding of phosphacan to Ng-CAM/L1, NCAM, and tenascin-C (FNIII domain) is mediated by complex-type N-linked oligosaccharides on the proteoglycan. Phosphacan, shows saturable, reversible, high-affinity binding to fibroblast growth factor-2 (FGF-2). The interaction is mediated primarily through the core protein. Immunocytochemical studies have also shown an overlapping localization of FGF-2 and phosphacan in the developing central nervous system. The core protein of phosphacan may also regulate the access of FGF-2 to cell surface signaling receptors in nervous tissue.
The carbonic anhydrase (CAH) domain of PTPζ has been shown to bind specifically to contactin. Contactin is a 140 kDa GPI membrane-anchored neuronal cell recognition protein expressed on the surface of neuronal cells. The CAH domain of RPTP zeta was shown to induce cell adhesion and neurite growth of primary tectal neurons, and differentiation of neuroblastoma cells. These responses were blocked by antibodies against contactin, demonstrating that contactin is a neuronal receptor for RPTP zeta. Caspr ((p190/Caspr, a contactin-associated transmembrane receptor) and contactin exist as a complex in rat brain and are bound to each other by means of lateral (cis) interactions in the plasma membrane. The extracellular domain of Caspr contains a neurophilin/coagulation factor homology domain, a region related to fibrinogen beta/gamma, epidermal growth factor-like repeats, neurexin motifs as well as unique PGY repeats found in a molluscan adhesive protein. The cytoplasmic domain of Caspr contains a proline-rich sequence capable of binding to a subclass of SH3 domains of signaling molecules. Caspr may function as a signaling component of contactin, enabling recruitment and activation of intracellular signaling pathways in neurons. The role of the extracellular domains in neural adhesion and neurite growth induction was investigated by the use of fusion protein constructs. The results suggested that binding of glial PTPζ to the contactin/Nr-CAM complex is important for neurite growth and neuronal differentiation.
PTPζ was shown to bind to a heparin-binding growth factor midkine through the chondroitin sulfate portion of the receptor. The interactions of pleiotrophin (PTN) with the receptor in U373-MG cells was also studied. Pleiotrophin was shown to bind to the spacer domain. Results suggested that PTN signals through “ligand-dependent receptor inactivation” of PTPζ and disrupts its normal roles in the regulation of steady-state tyrosine phosphorylation of downstream signaling molecules. PTN was shown to bind to and functionally inactivate the catalytic activity of PTPζ. An active site-containing domain of PTPζ both binds β-catenin and functionally reduces its levels of tyrosine phosphorylation when added to lysates of pervanadate-treated cells. In unstimulated cells, PTPζ was shown to be intrinsically active, and thought to function as an important regulator in the reciprocal control of the steady-state tyrosine phosphorylation levels of β-catenin by tyrosine kinases and phosphatases.
Using the yeast substrate-trapping system, several substrate candidates for PTPζ were isolated. The results indicated that GIT1/Cat-1 is a substrate molecule of PTPζ. In addition, PTPζ was shown to bind to the PSD-95/SAP90 family through the second phosphatase domain. Immunohistochemical analysis revealed that PTPζ and PSD-95/SAP90 are similarly distributed in the dendrites of pyramidal neurons of the hippocampus and neocortex. Subcellular fractionation experiments indicated that PTPζ is concentrated in the postsynaptic density fraction. These results suggested that PTPζ is involved in the regulation of synaptic function as postsynaptic macromolecular complexes with PSD-95/SAP90.
Voltage-gated sodium channels in brain neurons were also found to associate with the membrane bound forms of PTPζ and phosphacan. Both the extracellular domain and the intracellular catalytic domain of PTPζ interacted with sodium channels. Sodium channels were tyrosine phosphorylated and were modulated by the associated catalytic domains of PTPζ.